Rat leukemic basophil (2H3) cell line was stimulated to secrete histamine either with calcium-specific ionophores or by aggregation of plasma membrane receptors for IgE. The ionophore, A23187, at concentrations (less than 100 nM) well below those stimulating secretion elicited large increases in (Ca2+)i and at higher concentrations (200-1000nM) stimulated hydrolysis of membrane radiolabeled inositol phospholipids as well. The hydrolysis was dependent on the concentration of ionophore and presence of external Ca2+ and was correlated with the secretory response. The results pointed to generation of diacylglycerol rather than of inositol phosphates as a critical factor in the action of A23187. When secretory responses were plotted as a function of percent hydrolysis of inositol phospholipid, the curve was shifted leftwards in the presence of the phorbol ester, TPA, which, like diacylglycerol, is an activator of protein kinase C. Paradoxically, the increases in (Ca2+)i in response to both antigen and A23187 were highly dependent on intracellular ATP levels.